1. Field of the Invention
This invention concerns an improved process for isolating podophyllotoxin from crude preparations containing podophyllotoxin in combination with closely related analogs.
2. Description of Background Information
Podophyllotoxin is a well known and well characterized lignan. Two major review articles which describe its early isolation and characterization are PROGRESS IN THE CHEMISTRY OF ORGANIC NATURAL PRODUCTS, L. Zechmeister, ed., Springer Verlag, Vienna, 1958, "The Chemistry of Podophyllum," pp. 84-166; and Chemical Reviews 55:957-1068, "The Naturally Occurring Lignans," W.M. Hearon et al. These works describe how, in the late nineteenth century and early in this century, a number of studies show that resins obtained from American May apples (Podophyllum, pellatum L. (American podophyllum) contain a collection of interesting, structurally related, polycyclic lignan compounds, including podophyllotoxin, quercetin, and the like. See, for example, F.A. Thompson, Am. J. Pharm., 62:245 (1890); J.C. Umney, Pharm. J., 23:207 (1892); W.R. Dunstan et al., J. Chem. Soc., 73:209 (1898); W. Bosche et al., Ann., 494:126 (1932); and Spath et al., Ber., 65:1536 (1932).
With the passage of time, other plant sources of podophyllotoxin have been identified. Other studies have focused on the composition of the crude resins and have looked at the relationships existing among the numerous components. These studies have identified materials known as alpha- and beta-peltatin (J.L. Hartwell et al., J. Chem. Soc., 72:245 (1950)), demethylpodophyllotoxin, picropodophyllin, and picropodophylloglucoside (Nadkarni et al., J. Am. Chem. Soc. 75:1308 (1953)).
Much of the interest in podophyllotoxin and the crude podophyllum resin from which it is obtained stems from its long-recognized pharmacologic activity. The podophyllum resin is famous as an ancient American indian remedy with suggested uses for abnormal skin growth conditions such as warts and the like. Some of these uses were more formalized. For example, in 1942 crude podophyllum resin was suggested as a treatment of condyloma acuminatum, a type of venereal wart, by Kaplan, I.W., New Orleans Med. Surg. J. 94:388 (1942). Podophyllum resin has also been proposed for use in a wide range of skin diseases due to infectious agents, nonspecific dermatosises, metabolic diseases, benign growths, and malignant growths. In addition, the composition of crude podophyllum resin has been further reviewed to point out that, in addition to podophyllotoxin, the alpha- and beta-peltatins, and demethylpodophyllotoxin, it contains other materials such as desoxypodophyllotoxin, H. podophyllotoxinglucoside, H. picropodophyllinglucoside, alpha-peltatinglucoside, beta-peltatinglucoside, 4,-demethylpodophyllotoxinglucoside, dehydropodophyllotoxin, sikkimotoxin, quercetin, isormamnetin, quercetin-3-galactoside, and kaempferol. These compounds are all similar to each other in ring structure and the like. However, it is generally believed that podophyllotoxin itself is the most active, and therefore most desired, species.
This focus on podophyllotoxin has led to a great interest in obtaining it in a pure form. Certain characteristics of the mixture of compounds renders this difficult. For one, podophyllotoxin isomerizes to picropodophyllin when contacted with alkali. This isomerization is favored thermodynamically and therefore is not reversible. Picropodophyllin is inactive. Purification is further complicated by the close relationship of the many compounds.
Classic methods for isolation of podophyllotoxin have involved extraction followed by laborious purification (see, e.g., Borsche, Ann., noted above, Chem. Abstracts 26:3509 (1932); Dunstan, F.L., J. Chem. Soc., noted above; and Kursten, R., Arch. Pharm. 229:220-248 (1891)). In 1950 Hartwell and Detty (J. Am. Chem. Soc. 72:246 (1950)) disclosed processes in which podophyllum resin is extracted into chloroform, leaving a dark insoluble residue amounting to about 37%, or in which podophyllotoxin is precipitated from a solution of podophyllin in alcohol. The crude podophyllotoxin so isolated is then purified by chromatography on a column of alumina. In this chromatography this crude mixture is placed on the top of the column and eluted with alcohol benzene. Fractions are taken with the podophyllotoxin eluting earlier than beta- and alpha-peltatin. In this process, however, the beta-peltatin does elute directly after the podophyllotoxin, and the alpha-peltatin is eluted by a change in solvent.
Another method for purifying podophyllotoxin is shown in U.S. Pat. No. 4,680,399, entitled PROCESS FOR THE ISOLATION AND PURIFICATION OF PODOPHYLLOTOXIN. This patent focuses on the use of nonbenzene materials for a fractional crystallization and the use of base to remove impurities.
While each of these processes has been used in the past, they are not without their limitations, not the least of which has been low efficiencies which give rise to a relatively high cost to the podophyllotoxin itself. It is an object of this invention to provide a method for recovering podophyllotoxin from podophyllum resin which is of high efficiency and low cost.